Synthesis and cytotoxic evaluation of substituted sulfonyl-n- hydroxyguanidine derivatives as potential antitumor agents

Article abstract of DOI:10.1021/jm9607818

A series of sulfonyl-N-hydroxygnanidine derivatives was designed and synthesized for cytotoxic evaluation as potential anticancer agents on the basis of the lead compound LY-181984. Replacement of the ureido moiety of the lead compound with hydrexyguanidine provided a stable cytotoxic agent. The conformation of sulfonyl-N-hydroxyguanidine derivatives, such as N-(4- chlorophenyl-)-N-[(benzo[2,3]thiadiazol-4-yl)sulfonyl]-N-hydroxyguanidine (4g), investigated utilizing HMBC NMR, theoretical calculations, and X-ray crystallography, indicated stacking of the two aromatic rings. The derivatives were evaluated for in vitro cytoxicity against five human tumor cell lines, including HepG2, TSGH 8302, COLO 205, KB, and MOLT-4. The cytotoxic activities of the derived compounds against the human tumor cell lines were equal to or greater than that of the lead compound. N-(4- Chlorophenyl)-N'-[[3,5-dichloro-4-(4-nitrophenoxy)phenyl]sulfonyl].N_- hydroxyguanidine (4n) and N-(4-chlorophenyl)-N'-[[3,5-dichloro4-(2-chloro-4- nitrophenoxy)phenyl]sulfonyl]-N_-hydroxyguanidine (40) exhibited the greatest growth inhibition of solid tumor cell lines. Compound 40 was found to possess antitumor activity against murine K1735/M2 melanoma xenografts.

Full text of DOI:10.1021/jm9607818

2276
J . Med. Chem. 1997, 40, 2276-2286
Syn th esis a n d Cytotoxic Eva lu a tion of Su bstitu ted
Su lfon yl-N-h yd r oxygu a n id in e Der iva tives a s P oten tia l An titu m or Agen ts
J i-Wang Chern,* Yu-Ling Leu,† Shan-Shue Wang,‡ Ruwen J ou,‡ Chin-Fen Lee,‡ Pei-Chie Tsou,‡
Shih-Chung Hsu,‡ Yen-Chywan Liaw,§ and Hua-Mei Lin|
School of Pharmacy, College of Medicine, National Taiwan University, No. 1, Section 1, J en-Ai Road, Taipei (100), Taiwan,
Institute of Pharmacy, National Defense Medical Center, Taipei (100), Taiwan, Drug Development Division, Development
Center for Biotechnology, Hsi-Chih Cheng, Taipei Hsien, Taiwan, Institute of Molecular Biology, Academia Sinica, Taipei,
Taiwan, and Department of Pharmacy, Provincial Taoyuan Hospital, Taoyuan, Taiwan, Republic of China
Received November 12, 1996X
A series of sulfonyl-N-hydroxyguanidine derivatives was designed and synthesized for cytotoxic
evaluation as potential anticancer agents on the basis of the lead compound LY-181984.
Replacement of the ureido moiety of the lead compound with hydroxyguanidine provided a
stable cytotoxic agent. The conformation of sulfonyl-N-hydroxyguanidine derivatives, such as
N-(4-chlorophenyl)-N′-[(benzo[2,1,3]thiadiazol-4-yl)sulfonyl]-N′′-hydroxyguanidine (4g), inves-
tigated utilizing HMBC NMR, theoretical calculations, and X-ray crystallography, indicated
stacking of the two aromatic rings. The derivatives were evaluated for in vitro cytoxicity against
five human tumor cell lines, including HepG2, TSGH 8302, COLO 205, KB, and MOLT-4. The
cytotoxic activities of the derived compounds against the human tumor cell lines were equal to
or greater than that of the lead compound. N-(4-Chlorophenyl)-N′-[[3,5-dichloro-4-(4-nitro-
phenoxy)phenyl]sulfonyl]-N′′-hydroxyguanidine (4n ) and N-(4-chlorophenyl)-N′-[[3,5-dichloro-
4-(2-chloro-4-nitrophenoxy)phenyl]sulfonyl]-N′′-hydroxyguanidine (4o) exhibited the greatest
growth inhibition of solid tumor cell lines. Compound 4o was found to possess antitumor
activity against murine K1735/M2 melanoma xenografts.
In tr od u ction
is considered to combine the imino group of guanidine
with the hydroxylamino group of hydroxyurea, has been
reported to exhibit potent antiviral and anticancer
activities by inhibition of ribonucleotide reductase.19-22
On the basis of these precedents, sulfonyl-N-hydrox-
yguanidine derivatives such as compounds 4a -r can be
regarded as bioisosters of sulfonylurea, which may
possess increased stability. This paper herein describes
the synthesis and biological evaluation of compounds
4a -r as anticancer agents.
Sulfonylurea derivatives constitute an important class
of therapeutical agents in medicinal chemistry.1 More
recently, a series of sulfonylurea derivatives, including
LY 181984 (1) and LY186641 (2), was reported to
possess a broad spectrum of activity in several solid
tumor models,2-5 and one of these compounds, LY
186641, is in extensive clinical trials based on its
impressive preclinical activity and apparent lack of
toxicity to proliferating normal tissues.6-9 This series
of compounds were initially discovered by directly
utilizing in vivo tumor screening models to overcome
the traditionally poor correlation between cytotoxity and
antitumor activity.10-12 However, it is of considerable
interest that the mode of action of these compounds
differ from traditional anticancer drugs which typically
inhibit DNA, RNA, or protein synthesis.10 Since sulfo-
nylurea derivatives have been found to accumulate in
the cell mitochondria, the mitochondria may be the
target site for antitumor activity of these compounds.13-15
Sulfonylurea derivatives, however, are susceptible to
hydrolysis at physiological conditions.16 In a previous
communication of our synthetic studies of 1,2,4-ben-
zothiadiazine 1,1-dioxides, 2,10-dihydro-10-hydroxy-3H-
imidazo[1,2-b][1,2,4]benzothiadiazine 6,6-dioxide (3),
which contains a built-in sulfonylhydroxyguanidine
moiety, was found to exhibit activity against several
tumor cell lines, including KB, COLO 205, TSGH 8302,
and HepG2.17,18 Nevertheless, hydroxyguanidine, which
Ch em istr y
The target compounds indicated in Table 4 were
synthesized as outlined in Scheme 1. Starting with
sulfonyl chloride 5, amination and condensation of the
resulting sulfonamide derivatives 6 (Table 1) with
isothiocyanates were used for the preparation of the
thiourea derivatives 7 (Table 2). Sulfonylthioureas are
susceptible to nucleophilic attack. For example, these
compounds decomposed if recrystallized from alcohol.
Even when dissolved in DMSO-d6 for NMR spectro-
* Corresponding address: Prof. J i-Wang Chern, School of Pharmacy,
National Taiwan University, No. 1, Section 1, J en-Ai Road, Taipei
(100), Taiwan. Fax: 886-2-393-4221. Tel: 886-2-393-9462. chern@
jwc.mc.ntu.edu.tw.
† National Defense Medical Center.
‡ Development Center for Biotechnology.
§ Academia Sinica.
| Provincial Taoyuan Hospital.
X Abstract published in Advance ACS Abstracts, J une 1, 1997.
S0022-2623(96)00781-9 CCC: $14.00 © 1997 American Chemical Society

Sulfonyl-N-hydroxyguanidine Derivatives as Antitumor Agents
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 14 2277
Sch em e 1a
Ta ble 1. Chemical and Physical Properties of Sulfonamide
Derivatives 5a -p
a
(i) Liquid NH3, CH2Cl2, -78 °C, 10 min; (ii) (a) 1 N NaOH,
isothiocyanate, acetone, room temperature, 4-12 h; (b) 1 N HOAc;
(iii) (a) 1 N NaOH, MeI, acetone, room temperature, 30 min; (b) 1
N HOAc; (iv) NH2OH‚HCl, Et3N, CH3CN, 80-90 °C, 10-20 h or
DMF, room temperature, 2-3 days.
F igu r e 1. Three possible tautomeric forms of N,N′-disubsti-
tuted N′′-hydroxyguanidine.
scopic analysis, the compounds gradually decomposed,
in agreement with a previous report by J . E. Toth et
al.16 Therefore, compounds 7 was directly treated with
methyl iodide without isolation to afford the methyl-
pseudothiourea derivative 8 (Table 3), which was sub-
sequently reacted with hydroxylamine hydrochloride to
obtain the target compounds 4a -r (Table 4). The
guanidine derivatives 9 were also isolated as side
products, probably due to ammonia salt contamination
of the hydroxylamine hydrochloride. The low yield for
compounds 4a -r was due to the intensive column
chromatography to separate the compounds 4a -r and
9, which were close to each other in the column.
The sulfonyl-N-hydroxyguanidine moiety of compound
4 can be illustrated in three tautomeric forms (Figure
1). The preferred tautomeric form is A and was
1
elucidated from the following evidence. The H NMR
a
spectrum of 4g exhibited two D2O exchangeable proton
peaks at δ 9.40 and 9.74. The former peak corresponded
to one proton which was assigned to NH whereas the
latter peak was assigned to NH and OH protons. To
investigate the tautomeric state of this type of com-
pound, N-(4-chlorophenyl)-N′-(benzo[2,1,3]thiadiazol-4-
ylsulfonyl)-N′′-hydroxyguanidine (4g) was chosed as a
model compound. In the HMBC NMR spectrum of 4g,
the carbon signal (δ 125.0, C2 and C6) of the chloro-
phenyl group showed a 13C-1H long-range correlation
with the N-H signal (δ 9.47). However, the absence of
a long-range correlation between the N-H and the
benzo moiety of the benzo[2,1,3]thiadiazole ring pre-
sented their unambiguous identification. Therefore, the
HMBC NMR spectrum of 4g is consistent with tau-
tomers A and C, but not B.
Recrystallized from E (ethanol), EA (ethyl acetate), H (n-
hexane), M (methanol), and W (water).
angles of 4g are of interest to the issue of the tautom-
erism in 4g. By comparison of these values with those
of normal bases,23 it was concluded that these molecules
mostly adopt the A form in equilibrium with the other
minor forms shown in Figure 1. The sulfonyl group
together with the guanidine moiety adopts a planar
conformation with the torsion angle of N3-C7-N5-C8
being nearly 0° (-7°) and the C2, S1, N3, C7, N5, and
C8 atoms lying in a least-squares plane with mean
deviation of 0.3 Å. The plane of the guanidine moiety
is nearly perpendicular to the base plane whereas the
hydrophobic ring systems cluster together into hydro-
phobic pockets in the crystal lattices.
There are three possible tautomers of 4g. The bond
lengths of 4g in crystal, especially the bond distances
of C7-N3, C7-N4, and C7-N5, do not unambigiously
suggest that tautomer A is most favored. The differ-
ences in the total energy associated with these three
The structure of 4g was further investigated by X-ray
crystallography (Figure 2). The three-dimensional struc-
ture of 4g and lattice packing of 4g surprisingly reveals
base stacking (Figure 3). The bond distances and bond

2278 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 14
Chern et al.
Ta ble 2. Chemical, Physical Properties, and Cytotoxic Activities of N,N′-Disubstituted Thioureas 7a ,b,d ,g,j,k ,q,r
IC50 (µg/mL)a
COLO Hep-
yield,
%
TSGH MOLT-
no.
R
R1 mp, °C
formula
205
G2
KB 8302
4b
7a (4-methylphenyl)sulfonyl
7b phenylsulfonyl
H
H
H
H
H
H
172-173 22.6 C14H13ClN2O2S2
87
232 80
125
59
(E)c
165-168
(T)
7.2 C13H11ClN2O2S2
91
52
>300 66 >300
63
12
55
59
ND
39
65
33
7d (5-chloro-3-methylbenzo[b]thiophene-
2-yl)sulfonyl
7g benzo[2,1,3]thiadiazol-4-ylsulfonyl
188-189 91.0
C
C
C
C
C
C
16H12Cl2N2O2S3
57 61
124 63
216 66
214 196
120 41
62 25
55
42
(AN)
192-193 26.9
(AN)
156-157 44.3
(T/AC)
257-258 12.8
(C/T)
13H9ClN4O2S3
50
7j 5-(phenylsulfonyl)thiophene
17H12ClN2O4S4Na‚3/2H2O
17H13ClN2O4S4‚1/3H2O
14H11Cl2N2O2S2Na‚2H2O
16H10Cl3N2O2S3Na‚3/2H2O
65
79
7k [4-(phenylsulfonyl)thiophene-2-yl]sulfonyl
7q (4-methylphenyl)sulfonyl
220
45
217
55
Cl 252-253 88.0
(T/AC)
7r (5-chloro-3-methylbenzo[b]thiophene-
Cl 256
(T/AC)
80.7
57
59
2-yl)sulfonyl
1
83
>300 72
106
a
The cytotoxicity tests were replicated two times. Each treatment has three replications. The measurement of IC50 is described in
Materials and Methods. Percent inhibition in 20 µg/mL. c Recrystallized from AC (acetone), AN (acetonitrile), C (chloroform), E (ethyl
acetate), and T (toluene).
b
Ta ble 3. Chemical and Physical Properties of N,N′-Disubstituted S-Methylpseudothioureas 8a -r
no.
R
R1
H
mp, °C
yield, %
86.5
formula
8a
(4-methylphenyl)sulfonyl
phenylsulfonyl
173-174
(M)a
139-140
(M)
110
(M)
189-190
(E/C)
189-190
(M)
C15H15ClN2O2S2
8b
8c
8d
8e
8f
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
Cl
85
C14H13ClN2O2S2
[3,5-bis(trifluoromethyl)phenyl]sulfonyl
(5-chloro-3-methylbenzo[b]thiophene-2-yl)sulfonyl
benzo[b]thiophene-2-ylsulfonyl
98.4
72.8
91.0
48.7
83.7
94.9
55.7
75.9
43.1
81.1
95.3
99.6
89.7
85.6
96.9
99
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
16H11ClF6N2O2S2
17H14Cl2N2O2S3
16H13ClN2O2S3
16H13ClN2O3S2
14H11ClN4O2S3
17H14ClN3O2S3
15H12ClN3O3S3
18H15ClN2O4S4
18H15ClN2O4S4‚1/2H2O
21H15Cl2N3O3S2
20H15Cl2N3O5S2
20H14Cl3N3O5S2
20H13Cl4N3O5S2
18H21ClN2O2S2‚1/4H2O
15H14Cl2N2O2S2
17H13Cl3N2O2S3
benzofuran-2-ylsulfonyl
163-164
(M)
8g
8h
8i
benzo[2,1,3]thiadiazol-4-ylsulfonyl
216-217
(E/AC)
206-207
(E/M)
147
(2-pyrid-2-ylthiophene-5-yl)sulfonyl
(5-isoxazol-3-ylthiophene-5-yl)sulfonyl
5-(phenylsulfonyl)thiophene
(M)
8j
167-168
(E/C)
98-99
(M)
198
(E)
196
(M)
165
(M)
184
(M)
142
(M)
133-134
(M)
166-167
(E)
8k
8l
[4-(phenylsulfonyl)thiophene-2-yl]sulfonyl
[4-(3-chloro-2-cyanophenoxy)phenyl]sulfonyl
[4-(2-chloro-6-nitrophenoxy)phenyl]sulfonyl
[3,5-dichloro-4-(4-nitrophenoxy)phenyl]sulfonyl
[3,5-dichloro-4-(2-chloro-4-nitrophenoxy)phenyl]sulfonyl
(4-n-butoxyphenyl)sulfonyl
8m
8n
8o
8p
8q
8r
(4-methylphenyl)sulfonyl
(5-chloro-3-methylbenzo[b]thiophene-2-yl)sulfonyl
a
Recrystallized from AC (acetone), C (chloroform), E (ethanol), and M (methanol).

Sulfonyl-N-hydroxyguanidine Derivatives as Antitumor Agents
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 14 2279
Ta ble 4. Chemical, Physical Properties, and Cytotoxic Activities of N,N′-Disubstituted Sulfonyl-N-hydroxyquanidines 4a -r against
Human Tumor Cell Lines
IC50 (µg/mL)
yield,
%
COLO Hep-
TSGH MOLT-
8302
no.
R
R1
H
mp, °C
formula
205a
58
G2
KB
46 46
4
4a (4-methylphenyl)sulfonyl
4b phenylsulfonyl
201-203
(M)b
200-201
(M)
12.7 C14H14ClN3O3S
27.2 C13H12ClN3O3S
49
6
7
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
53
58
60
80
54
233
140
176
46 52
57 66
29 44
55 76
67 ND
35 93
61 208
55 149
29 41
10 37
39 168
83 53
4c [3,5-bis(trifluoromethyl)phenyl]sulfonyl
192
21.9
40.9
27.6
63.9
31.2
52.2
37.5
11.6
19.8
44.1
35.2
23.9
30.6
34.3
24.9
24.8
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
15H10ClF6N3O3S
16H13Cl2N3O3S
15H12ClN3O3S2
15H12ClN3O4S
13H10ClN5O3S2
16H13ClN4O3S2
14H11ClN4O4S2
17H14ClN3O5S3
17H14ClN3O5S3
20H14Cl2N4O4S
19H14Cl2N4O6S
19H13Cl3N4O6S
19H12Cl4N4O6S‚H2O
17H20ClN3O3S
14H13Cl2N3O3S
16H12Cl3N3O3S2
44
38
57
ND
33
75
9
(M/W)
245-247
(M)
264-266
(M)
207-209
(M)
217-218
(M)
210(dec)
(AN)
184
(T)
195-196
(M)
195(dec)
(C)
199(dec)
(M)
199
(AN)
212-214
(M)
180
(C)
4d (5-chloro-3-methylbenzo[b]thiophene-
2-yl)sulfonyl
4e benzo[b]thiophene-2-ylsulfonyl
4f benzofuran-2-ylsulfonyl
>200 >200
4g benzo[2,1,3]thiadiazol-4-ylsulfonyl
4h (2-pyrid-2-ylthiophene-5-yl)sulfonyl
87
74
45
121
4i
4j
(5-isoxazol-3-ylthiophene-5-yl)sulfonyl
5-(phenylsulfonyl)thiophene
52 >300
59
45
92
ND
22
95
ND
55
56
18
7
4k [4-(phenylsulfonyl)thiophene-2-yl]sulfonyl
4l [4-(3-chloro-2-cyanophenoxy)phenyl]sulfonyl
175
178 >300
4m [4-(2-chloro-6-nitrophenoxy)phenyl]sulfonyl
>100 >100
4n [3,5-dichloro-4-(4-nitrophenoxy)phenyl]-
sulfonyl
4o [3,5-dichloro-4-(2-chloro-4-nitrophenoxy)-
phenyl]sulfonyl
12
6
22
49
63
72
12
7
7
7
4p (4-n-butoxyphenyl)sulfonyl
175-176
(M)
49
10
50 37
67
4q (4-methylphenyl)sulfonyl
Cl 195(dec)
(M)
Cl 232-233
8
4r (5-chloro-3-methylbenzo[b]thiophene-
>300 >300 >300 >300
>300
2-yl)sulfonyl
(AN/E/EA)
a
The cytotoxicity tests were replicated two times. Each treatment has three replications. The measurement of IC50 is described in
b
Materials and Methods. Recrystallized from AC (acetone), AN (acetonitrile), C (chloroform), E (ethanol), EA (ethyl acetate), M (methanol),
T (toluene), and W (water).
getically favored, the bond distances of C7-N3, C7-
N4, and C7-N5 are not equal to either pure C-N single
or double bond lengths. This suggests some degree of
coexistence of the three forms and/or some degree of
delocalization among these bonds. However, the clear
location of the proton density around N5 with correct
geometry in the X-ray crystallography (Figure 2) indi-
cates that tautomer A predominates in crystals. Hence,
on the basis of the HMBC NMR spectrum and X-ray
crystallograph, studies illustrated that tautomer A is
overall most favored in this type of compound.
Resu lts a n d Discu ssion
The sulfonylthioureas 7a ,b,d ,g,j,k ,q,r and sulfonyl-
N-hydroxyguanidine derivatives 4a -r were evaluated
by the MTT assay for in vitro cytotoxicity against five
human tumor cell lines, including human hepatocellular
carcinoma (HepG2), human epidermoid cervical carci-
noma (TSGH 8302), human colon adenocarcinoma
(COLO 205), human epidermoid oral carcinoma (KB),
and human acute lymphoblastic leukemia (MOLT-4).
The human COLO 205, HepG2, KB, and TSGH 8302
cell lines were employed as a small panel for the
F igu r e 2. ORTEP drawing of N-(4-chlorophenyl)-N′-(benzo-
[2,1,3]thiadiazol-4-ylsulfonyl)-N′′-hydroxyguanidine (4g).
tautomers are 4.3 kcal/mol between tautomers A and
B and 1.8 kcal/mol between tautomers A and C, based
on the calculation using MOPAC24 with PM3 force field
parameters.25 Although tautomer A is the most ener-

2280 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 14
Chern et al.
Ta ble 5. Antitumor Activity of LY181984 and 4o against
Murine K1735/M2 Melanoma Xenografta
body
treatment weight
dose
administered schedule change TGI
agent
(mg/kg)
route
(day)
(g)
(%)
control
LY181984 300
4o 300
vehicle only
po
po
po
5-9, 12-16
3.0
5-9, 12-16 -1.2 64.7
5-9, 12-16
1.3 70.7
a
Tumor growth inhibition (TGI %) was determined at day 20
after tumor transplantation. Body weight change (gm/mice) was
calculated from day 0 to day 20. The TGI % and tumor weight
were estimated as described in Materials and Methods.
F igu r e 3. The lattice packing diagram of N-(4-chlorophenyl)-
N′-[(benzo[2,1,3]thiadiazol-4-ylsulfonyl)-N′′-hydroxyguani-
dine (4g).
screening of new antisolid tumor agents whereas
MOLT-4 was used as a representative human blood cell.
The criteria for selection of an agent for further in vivo
investigation of drug efficacy was a demonstration of
greater activity against COLO 205, HepG2, KB, and
TSGH 8302 cells compared to MOLT-4 cells in the in
vitro cytotoxicity assay.
The cytotoxic activities of these compounds are pre-
sented in Table 2 and 4. Although the thiourea deriva-
tives 7a ,b,d ,g,j,k ,q,r were as active as LY 181984, they
were as subject to nucleophilic attack as compound 1.
As shown in Table 4, replacement of the urea moiety of
the lead compound LY 181984 with N-hydroxyguanidine
produced compounds 4a -c with similar cytotoxicity as
LY-181984 in all cell lines. Compounds 4a ,b were more
active against HepG2 and Molt-4 than LY181984
whereas compounds 4d -k were as active as 4a . This
indicates that the aromatic ring attached to the sulfonyl
moiety of 4a can be substituted with different hetero-
cycles such as benzothiophene, benzofuran, benzo[2,1,3]-
thiadiazole, and thiophene without substantially affect-
ing the general cytotoxicity of this class of compounds.
Introduction of a butyloxy group (4p ) at the para
position of the phenylsulfonyl group of 4b also retained
the activity, indicating that the para position of the
phenylsulfonyl moiety can tolerate a bulky substituent.
However, replacement of the butyloxy moiety with a
phenoxy, such as 4l and 4m , dramatically reduced
activity. Activity, however, was significantly enhanced
in compounds 4n and 4o, in which two chloro atoms
were introduced at the 3′- and 5′-positions of the
attached phenylsulfonyl moiety.
F igu r e 4. Tumor growth curves of murine K-1735/M2 mela-
noma xenograft treated with LY181984 and 4o. Five days
following tumor transplantation, mice were treated po with a
daily dose of 300 mg/kg LY181984 or 4o for two cycles of 5
consecutive days [(Q1DX5)2] at day 5 and day 12. Control mice
were administered po with 2.5% cremophor. Each point, mean
tumor weight (mg), were from five animals/group: (b) control,
(2) LY181984, ([) 4o.
starting on days 5 and 12. The tumor growth inhibition
(TGI %) of LY-181984 and 4o were 64.7% and 70.7%,
respectively, 20 days after tumor transplantation (Table
5). Both LY-181984 and 4o delayed the growth of solid
melanoma tumor in the animal model (Figure 4). The
mean body weight of mice increased 3 g in the control
group and increased 1.3 g in the 4o treatment group,
but decreased 1.2 g in the LY-181984 treatment group,
suggesting that 4o was less toxic than LY-181984.
In summary, new N-hydroxyguanidine derivatives
were synthesized via a bioisosteric displacement of the
ureido moiety of LY 181984 with hydroxyguanidine.
These molecules are chemically stable and displayed
potent inhibitory properties against several solid tumor
lines in vitro. No pharmacological mechanism has yet
been determined to explain these effects.
Compound 4o exhibited enhanced activity against
COLO 205 (IC50, 6.32 µg/mL), KB (IC50, 6.70 µg/mL),
and TSGH 8302 (IC50, 7.15 µg/mL) cells compared with
that of MOLT-4 cells (IC50, 55.88 µg/mL). Compound
4o was therefore selected for further drug development.
The activity of 4o against solid tumors was examined
in a murine K-1735/M2 melanoma xenograft model. LY-
181984 or 4o was given orally with a daily dose of 300
mg/kg for two 5-consecutive-day periods [(qid × 5)2]
Exp er im en ta l Section
Gen er a l Meth od s. Melting points were obtained on an
Electrothermal apparatus and are uncorrected. 1H and 13C
nuclear magnetic resonance spectra were recorded either on
a J EOL J NM-EX400 spectrometer at the National Taiwan
Normal University or on a Bruker Model AM 300 spectrometer
at the National Taiwan University, Taipei, and are reported
in parts per million with DMSO-d6 as internal standard on a

Sulfonyl-N-hydroxyguanidine Derivatives as Antitumor Agents
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 14 2281
δ scale. EI mass spectra were recorded on a J EOL J MS-D100
mass spectrometer at the National Taiwan University. El-
emental analyses for C, H, and N were carried out either on a
Heraeus elemental analyzer at the Cheng-Kong University,
Tainan, or on a Perkin-Elmer 240 elemental analyzer in the
National Taiwan University, Taipei, and were within (0.4%
of the theoretical values.
ArH), 8.57 (dd, J ) 5.0 Hz, J ) 1.7 Hz, 1H, ArH); 13C NMR
(75 MHz, DMSO-d6) δ 119.6, 124.0, 125.0, 131.2, 137.9, 146.9,
149.1, 150.0, 151.0. Anal. (C9H8N2O2S2) C, H, N.
5-Isoxa zol-3-ylth iop h en e-2-su lfon a m id e (6i): MS m/z
230 (M+); 1H NMR (300 MHz, DMSO-d6) δ 7.06 (s, 1H,
isoxazole-H), 7.62 (d, J ) 4.2 Hz, 1H, thiophene-H), 7.69 (d, J
) 4.2 Hz, 1H, thiophene-H), 7.89 (s, 2H, NH2), 8.70 (s, 1H,
isoxzole-H); 13C NMR (75 MHz, DMSO-d6) δ 101.3, 127.5,
130.8, 131.7, 147.5, 152.0, 162.0. Anal. (C7H6N2O3S2) C, H,
N.
P r ep a r a t ion of Su lfon a m id es (6a -d ,g-p ): Gen er a l
P r oced u r e. Liquid ammonia (20 mL) was added to a solution
containing appropriate sulfonyl chloride (10 g, 52.45 mmol)
in dichloromethane (100 mL) at -78 °C. After the mixture
was stirred at -78 °C for 4 h, precipitates were removed by
filtration and the filtrate was concentrated in vacuo to remove
the solvent. The residue was then recrystallized from the
appropriate solvent to give the desired compounds. The mp
and yield data are summarized in Table 1. The analytical data
are given below.
2-(P h en ylsu lfon yl)th iop h en e-5-su lfon a m id e (6j): MS
1
m/z 303 (M
+); H NMR (300 MHz, DMSO-d6) δ 7.60 (d, J )
3.8 Hz, 1H, thiophene-H), 7.68 (t, J ) 7.3 Hz, 2H, ArH), 7.77
(t, J ) 7.3 Hz, 1H, ArH), 7.88 (d, J ) 3.8 Hz, 1H, thiophene-
H), 8.02 (s, 2H, NH2), 8.02 (d, J ) 7.3 Hz, 2H, ArH); 13C NMR
(75 MHz, DMSO-d6) δ 127.7, 130.5, 130.8, 134.9, 140.8, 145.7,
153.4. Anal. (C10H9NO4S3) C, H, N.
1
4-Tolu en esu lfon a m id e (6a ): MS m/z 171 (M+); H NMR
4-(P h en ylsu lfon yl)th iop h en e-2-su lfon a m id e (6k ): MS
m/z 303 (M+); 1H NMR (300 MHz, DMSO-d6) δ 7.65-7.77 (m,
4H, ArH), 7.88 (s, 2H, NH2), 8.02 (d, J ) 7.1 Hz, 2H, ArH),
8.70 (d, J ) 1.6 Hz, 1H, thiophene-H); 13C NMR (75 MHz,
DMSO-d6) δ 127.4, 127.7, 127.7, 130.4, 134.6, 137.0, 140.8,
149.5. Anal. (C10H9NO4S3) C, H, N.
(300 MHz, CDCl3) δ 2.44 (s, 3H, CH3), 4.89 (s, 2H, NH2), 7.32
(d, J ) 8.2 Hz, 2H, ArH), 7.82 (d, J ) 8.2 Hz, 2H, ArH).
Ben zen esu lfon a m id e (6b): MS m/z 158 (M+); 1H NMR
(300 MHz, CDCl3) δ 4.92 (s, 2H, NH2), 7.60-7.51 (m, 3H, ArH),
7.96-7.92 (m, 2H, ArH).
3,5-Bis(tr iflu or om eth yl)ben zen esu lfon a m id e (6c): MS
m/z 293 (M+); 1H NMR (300 MHz, DMSO-d6) δ 7.76 (s, 2H,
NH2), 8.40 (s, 2H, ArH), 8.44 (s, 1H, ArH); 13C NMR (75 MHz,
DMSO-d6) δ 122.6 (q, J ) 270 Hz, CF3), 125.8, 126.4, 131.2 (q,
J ) 34 Hz, CCF3), 146.5. Anal. (C8H5F6NO2S) C, H, N.
5-Ch lor o-3-m et h ylb en zo[b]t h iop h en e-2-su lfon a m id e
(6d ): MS m/z 261 (M+); 1H NMR (400 MHz, DMSO-d6) δ 2.60
(s, 3H, CH3), 7.53 (dd, J ) 8.7 Hz, J ) 2.0 Hz, 1H, ArH), 7.88
(s, 2H, NH2), 7.99 (d, J ) 2.0 Hz, 1H, ArH), 8.06 (d, J ) 8.7
Hz, 1H, ArH); 13C NMR (75 MHz, DMSO-d6) δ 12.3, 123.5,
125.1, 127.5, 130.7, 133.9, 136.7, 141.3, 141.9. Anal. (C9H8-
ClO2S2) C, H, N.
4-(3-Ch lor o-2-cya n op h en oxy)ben zen esu lfon a m id e (6l):
1
MS m/z 308 (M+ - 1); H NMR (300 MHz, DMSO-d6) δ 7.11
(d, J ) 8.3 Hz, 1H, ArH), 7.36 (d, J ) 8.7 Hz, 2H, ArH), 7.41
(s, 2H, NH2), 7.57 (d, J ) 8.2 Hz, 1H, ArH), 7.73 (t, J ) 8.3
Hz, 1H, ArH), 7.90 (d, J ) 8.7 Hz, 2H, ArH); 13C NMR (75
MHz, DMSO-d6) δ 106.2, 113.4, 118.0, 119.6, 125.7, 128.7,
136.5, 137.1, 141.0, 157.7, 159.4. Anal. (C13H9ClN2O3S) C, H,
N.
4-(2-Ch lor o-6-n itr oph en oxy)ben zen esu lfon a m id e (6m ):
1
MS m/z 229 (M+); H NMR (300 MHz, DMSO-d6) δ 7.07 (d, J
) 8.7 Hz, 2H, ArH), 7.32 (s, 2H, NH2), 7.63 (t, J ) 8.2 Hz, 1H,
nitrophenoxy-H), 7.81 (d, J ) 8.7 Hz, 2H, ArH), 8.07 (dd, J )
8.0 Hz, J ) 1.0 Hz, 1H, nitrophenoxy-H), 8.19-8.15 (m, 1H,
nitrophenoxy-H); 13C NMR (75 MHz, DMSO-d6) δ 115.2, 125.0,
127.9, 128.1, 129.2, 136.0, 138.8, 142.2, 144.3, 158.6. Anal.
(C12H9ClN2O5S) C, H, N.
Ben zo[b]th iop h en e-2-su lfon a m id e (6e): To a solution of
benzothiophene (2.23 g, 16.6 mmol) in THF (40 mL) at room
temperature was added 1.6 M n-butyllithium in hexane (10.4
mL, 16.6 mmol). The reaction mixture was refluxed for 4 h
and then evaporated to dryness in vacuo. To the residue were
added water (100 mL), sodium acetate (10.89 g, 0.13 mol), and
hydroxylamine O-sulfonic acid (6.26 g, 0.05 mol). The mixture
was allowed to stir at room temperature for 8 h and was then
ether extracted (75 mL × 2). The organic layer was extracted
with 1 N sodium hydroxide (50 mL × 3). The aqueous layer
was collected and neutralized with 1 N hydrochloride solution
followed by extraction with dichloromethane (50 mL × 3). The
organic layer was collected and dried over anhydrous sodium
sulfate. Concentration in vacuo yielded a yellow solid which
was recrystallized from methanol and water (1:1) to give 6e
3,5-Dich lor o-4-(4-n it r op h en oxy)b en zen esu lfon a m id e
(6n ): MS m/z 362 (M+); 1H NMR (300 MHz, DMSO-d6) δ 7.15
(d, J ) 9.1 Hz, 2H, ArH), 7.71 (s, 2H, NH2), 8.05 (s, 2H, ArH),
8.26 (d, J ) 9.1 Hz, 2H, ArH); 13C NMR (75 MHz, DMSO-d6)
δ 115.7, 126.4, 127.1, 129.1, 143.1, 147.6, 160.2. Anal. (C12H8-
Cl2N O S) C, H, N.
2
5
4-(2-Ch lor o-4-n it r op h e n oxy)-3,5-d ich lor ob e n ze n e -
su lfon a m id e (6o): MS m/z 229 (M+); 1H NMR (300 MHz,
DMSO-d6) δ 6.95 (d, J ) 9.2 Hz, 1H, ArH), 7.72 (s, 2H, NH2),
8.13 (dd, J ) 9.0 Hz, J ) 3.1 Hz, 2H, ArH), 8.52 (d, J ) 3.2
Hz, 1H, ArH); 13C NMR (75 MHz, DMSO-d6) δ 114.8, 122.0,
124.6, 126.5, 127.1, 128.8, 143.2, 144.0, 147.4, 155.5. Anal.
(C12H7Cl3N2O5S) C, H, N.
1
(0.28 g, 7.8%): mp 210-211 °C; MS m/z 213 (M+); H NMR
(300 MHz, DMSO-d6) δ 7.46-7.54 (m, 2H, ArH), 7.87 (s, 2H,
NH2), 7.92 (s, 1H, ArH), 8.00-8.09 (m, 2H, ArH); 13C NMR
(75 MHz, DMSO-d6) δ 123.4, 125.8, 126.0, 127.1, 127.3, 138.0,
140.6, 146.2. Anal. (C8H7NO2S2) C, H, N.
4-n -Bu toxyben zen esu lfon a m id e (6p ): MS m/z 362 (M+);
1H NMR (300 MHz, DMSO-d6) δ 0.92 (t, J ) 7.3 Hz, 3H, CH3),
1.49-1.36 (m, 2H, CH2), 1.75-1.64 (m, 2H, CH2), 4.03 (t, J )
6.4 Hz, 2H, OCH2), 7.06 (d, J ) 8.6 Hz, 2H, ArH), 7.16 (s, 2H,
NH2), 7.72 (d, J ) 8.7 Hz, 2H, ArH); 13C NMR (75 MHz,
DMSO-d6) δ 13.6, 18.6, 30.5, 67.6, 114.4, 127.6, 136.0, 161.0.
Anal. (C10H15NO3S) C, H, N.
P r ep a r a tion of N,N′-Disu bstitu ted Th iou r ea s 7a ,b,d ,-
g,j,k ,q,r : Gen er a l P r oced u r e. Sodium hydroxide solution
(1 N, 1 mL, 1 mmol) was added to a solution containing the
appropriate sulfonamide (1.0 mmol) in acetone (25 mL). After
the mixture was stirred at room temperature for 30 min, the
appropriate isothiocyanate (1.0 mmol) was added. After 4 h
of reflux, the mixture was cooled to room temperature and
neutralized with 1 N acetic acid solution to pH 5. The mixture
was allowed to stir at room temperature for 30 min before
water (45 mL) was added to produce a white precipitate which
was collected by filtration. After the solid was dried in the
oven, it was recrystallized from the appropriate solvent to give
the desired compound. The mp and yield data are summarized
in Table 2. The analytical data are given below.
Ben zofu r a n -2-su lfon a m id e (6f) was prepared in 10.2%
yield starting from benzofuran following the same procedures
as for 6e. An analytical sample was prepared by recrystalli-
zation from water and methanol (4:1): mp 160-161 °C; MS
m/z 197 (M+); 1H NMR (300 MHz, DMSO-d6) δ 7.36-7.41 (m,
1H, ArH), 7.45 (d, J ) 1.1 Hz, 1H, furan-H), 7.48-7.54 (m,
1H, ArH), 7.71 (d, J ) 7.9 Hz, 1H, ArH), 7.80 (d, J ) 7.3 Hz,
1H, ArH), 8.01 (s, 2H, NH2); 13C NMR (75 MHz, DMSO-d6) δ
109.4, 112.3, 123.5, 124.6, 127.7, 154.9. Anal. (C8H7NO3S)
C, H, N.
Ben zo[2,1,3]th ia d ia zole-4-su lfon a m id e (6g): MS m/z 215
(M+); 1H NMR (300 MHz, DMSO-d6) δ 7.65 (s, 2H, NH2), 7.86
(dd, J ) 8.7 Hz, J ) 7.1 Hz, 1H, ArH), 8.19 (d, J ) 7.1 Hz, 1H,
ArH), 8.36 (d, J ) 8.7 Hz, 1H, ArH); 13C NMR (75 MHz,
DMSO-d6) δ 125.4, 128.7, 128.9, 135.0, 148.8, 155.0. Anal.
(C6H5N3O2S2‚1/2H2O) C, H, N.
2-P yr id -2-ylth iop h en e-5-su lfon a m id e (6h ): MS m/z 240
(M+); 1H NMR (300 MHz, DMSO-d6) δ 7.37 (dd, J ) 7.2 Hz, J
) 5.0 Hz, 1H, ArH), 7.56 (d, J ) 3.8 Hz, 1H, thiophene-H),
7.73 (s, 2H, NH2), 7.79 (d, J ) 3.8 Hz, 1H, thiophene-H), 7.89
(td, J ) 7.3 Hz, J ) 1.7 Hz, 1H, ArH), 8.01 (d, J ) 7.4 Hz, 1H,
N-[(4-Meth ylp h en yl)su lfon yl]-N′-(4-ch lor op h en yl)th io-
u r ea (7a ): MS m/z 341 (M+ + 1); 1H NMR (400 MHz, DMSO-
d6) δ 2.40 (s, 3H, CH3), 7.37-7.48 (m, 6H, ArH), 7.82 (d, J )

2282 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 14
Chern et al.
7.8 Hz, 2H, ArH), 10.22 (brs, 1H, NH); 13C NMR (75 MHz,
DMSO-d6) δ 21.5, 126.1, 126.5, 128.1, 128.9, 129.2, 129.8,
137.7, 144.3, 178.3. Anal. (C14H13ClN2O2S2) C, H, N.
N-(P h en ylsu lfon yl)-N′-(4-ch lor op h en yl)th iou r ea (7b):
N -(P h e n ylsu lfon yl)-N ′-(4-ch lor op h e n yl)-S -m e t h yl-
1
p seu d oth iou r ea (8b): MS m/z 343 (M+ + 2); H NMR (300
MHz, DMSO-d6) δ 2.47 (s, 3H, SCH3), 7.36 (d, J ) 8.7 Hz, 2H,
ArH), 7.45 (d, J ) 8.7 Hz, 2H, ArH), 7.53-7.66 (m, 3H, ArH),
7.86 (d, J ) 8.4 Hz, 2H, ArH), 9.71 (s, 1H, NH); 13C NMR (100
MHz, DMSO-d6) δ 14.8, 126.2, 127.2, 128.7, 128.9, 131.3, 132.2,
136.3, 142.3, 166.3. Anal. (C14H13ClN2O2S2) C, H, N.
N-(4-Ch lor oph en yl)-N′-[[3,5-bis(tr iflu or om eth yl)ph en yl]-
su lfon yl]-S-m eth ylp seu d oth iou r ea (8c): MS m/z 477 (M+);
1H NMR (300 MHz, DMSO-d6) δ 2.56 (s, 3H, SCH3), 7.35 (d, J
) 8.7 Hz, 2H, ArH), 7.45 (d, J ) 8.7 Hz, 2H, ArH), 8.38 (s,
2H, ArH), 8.45 (s, 1H, ArH), 10.04 (s, 1H, NH); 13C NMR (75
MHz, DMSO-d6) δ 15.4, 123.0 (q, J ) 271 Hz, CF3), 126.5,
126.7, 127.2, 128.2, 129.2, 131.5 (q, J ) 33.9 Hz, CCF3), 136.6,
145.5, 167.7. Anal. (C16H11F6ClN2O2S2) C, H, N.
1
MS m/z 295 (M+ - 32); H NMR (300 MHz, DMSO-d6) δ 7.39
(d, J ) 8.1 Hz, 2H, ArH), 7.47 (d, J ) 8.4 Hz, 2H, ArH), 8.17-
7.62 (m, 3H, ArH), 7.94 (d, J ) 8.1 Hz, 2H, ArH), 10.30 (s,
1H, NH). Anal. (C13H11ClN2O2S2) C, H, N.
N-(4-Ch lor op h en yl)-N′-[(5-ch lor o-3-m et h ylb en zo[b]-
th iop h en e-2-yl)su lfon yl]th iou r ea (7d ): MS m/z 398 (M+
-
1
33); H NMR (300 MHz, DMSO-d6) δ 2.63 (s, 3H, CH3), 7.36
(d, J ) 8.8 Hz, 2H, ArH), 7.56 (d, J ) 8.8 Hz, 3H, ArH), 8.03
(d, J ) 1.9 Hz, 1H, ArH), 8.08 (d, J ) 8.7 Hz, 1H, ArH), 10.15
(brs, 1H, NH); 13C NMR (75 MHz, DMSO-d6) δ 12.6, 121.2,
123.7, 124.1, 125.0, 125.1, 127.4, 127.6, 128.7, 129.1, 130.6,
138.0, 140.7. Anal. (C16H12Cl2N2O2S3) C, H, N.
N-(4-Ch lor op h en yl)-N′-[(5-ch lor o-3-m et h ylb en zo[b]-
th iop h en e-2-yl]su lfon yl]-S-m eth ylp seu d oth iou r ea (8d ):
N-(4-Ch lor op h en yl)-N′-(ben zo[2,1,3]th ia d ia zol-4-yl)su l-
1
MS m/z 446 (M+ + 1); H NMR (300 MHz, DMSO-d6) δ 2.56
1
fon yl)th iou r ea (7g): MS m/z 350 (M+ - 35); H NMR (300
(s, 3H, CH3), 2.57 (s, 3H, SCH3) 7.40-7.50 (AB q, J ) 8.9 Hz,
4H, ArH), 7.55 (dd, J ) 8.6 Hz, 2.0 Hz, 1H, ArH), 8.00 (d, J )
2.0 Hz, 1H, ArH), 8.07 (d, J ) 8.6 Hz, 1H, ArH); 13C NMR (75
MHz, DMSO-d6) δ 12.4, 15.4, 123.6, 125.0, 127.5, 127.6, 129.2,
130.7, 131.5, 134.7, 136.6, 137.1, 141.1, 167.3, 167.4. Anal.
(C17H14Cl2N2O2S3) C, H, N.
MHz, DMSO-d6) δ 7.36 (d, J ) 8.8 Hz, 2H, ArH), 7.51 (d, J )
8.8 Hz, 2H, ArH), 7.91 (dd, J ) 8.8 Hz, 7.1 Hz, ArH), 8.36 (d,
J ) 7.1 Hz, 1H, ArH), 8.43 (d, J ) 8.8 Hz, 1H, ArH), 10.18
(brs, 1H, NH); 13C NMR (75 MHz, DMSO-d6) δ 120.9, 125.0,
125.8, 127.4, 128.7, 129.0, 129.2, 129.4, 133.1, 155.2. Anal.
(C13H9ClN4O2S3) C, H, N.
N -(4-Ch lor op h e n yl)-N ′-(b e n zo[b]t h iop h e n e -2-ylsu l-
fon yl)-S-m eth ylp seu d oth iou r ea (8e): MS m/z 398 (M+); 1H
NMR (300 MHz, DMSO-d6) δ 2.55 (s, 3H, SCH3), 7.40-7.55
(m, 6H, ArH), 8.00-8.08 (m, 3H, ArH), 9.86 (s, 1H, NH); 13C
NMR (75 MHz, DMSO-d6) δ 35.2, 106.6, 123.3, 123.4, 125.80,
126.2, 127.4, 127.7, 128.2, 129.2, 136.6, 137.7, 141.1, 143.9.
Anal. (C16H13ClN2O2S3) C, H, N.
N-(4-Ch lor op h en yl)-N′-[[2-(p h en ylsu lfon yl)th iop h en e-
5-yl]su lfon yl]th iou r ea (7j): MS m/z 473 (M+); 1H NMR (300
MHz, DMSO-d6) δ 7.21 (d, J ) 9.0 Hz, 2H, ArH), 7.46 (d, J )
7.2 Hz, 1H, ArH), 7.63-7.73 (m, 7H, ArH), 7.99 (d, J ) 7.2
Hz, 2H, ArH), 9.51 (brs, 1H, NH); 13C NMR (75 MHz, DMSO-
d6) δ 117.1, 122.3, 125.5, 127.3, 127.4, 128.2, 130.0, 130.3,
132.7, 134.4, 140.2, 143.4. Anal. (C17H13ClN2O4S4‚3/2H2O) C,
H, N.
N-(4-Ch lor op h en yl)-N′-[[2-(p h en ylsu lfon yl)th iop h en e-
4-yl]su lfon yl]th iou r ea (7k ): MS m/z 351 (M+ - 125); 1H
NMR (300 MHz, DMSO-d6) δ 7.14 (d, J ) 8.9 Hz, 2H, ArH),
7.46 (d, J ) 8.9 Hz, 2H, ArH), 7.54 (d, J ) 1.6 Hz, 1 H,
thiophene-H), 7.61-7.74 (m, 3H, ArH), 7.94-7.97 (m, 2H,
ArH), 8.40 (d, J ) 1.6 Hz, 1H, thiophene-H), 8.67 (brs, 2H,
NH). Anal. (C17H13ClN2O4S4‚1/3H2O) C, H, N.
N-(3,4-Dich lor oph en yl)-N′-(4-tolylsu lfon yl)th iou r ea (7q):
MS m/z 375 (M+); 1H NMR (300 MHz, DMSO-d6) δ 2.38 (s,
3H, CH3), 7.37 (d, J ) 8.0 Hz, 2H, Ar-H), 7.45 (dd, J ) 8.8 Hz,
J ) 2.3 Hz, 1H, Ar-H), 7.54 (d, J ) 8.8 Hz, 1H, Ar-H), 7.79 (d,
J ) 8.2 Hz, 2H, Ar-H), 7.90 (s, 1H, Ar-H), 10.10 (brs, 1H, Ar-
H); 13C NMR (75 MHz, DMSO-d6) δ 21.4, 123.5, 124.6, 126.0,
128.1, 128.2, 129.5, 129.7, 130.6, 130.9, 139.6, 179.4. Anal.
(C14H11Cl2N2O2S2Na‚2H2O) C, H, N.
N-(4-Ch lor oph en yl)-N′-(ben zofu r an -2-ylsu lfon yl)-S-m e-
1
th ylp seu d oth iou r ea (8f): MS m/z 383 (M+); H NMR (300
MHz, DMSO-d6) δ 2.56 (s, 3H, SCH3), 7.36-7.57 (m, 7H, ArH),
7.73-7.81 (m, 2H, ArH), 9.95 (s, 1H, NH); 13C NMR (75 MHz,
DMSO-d6) δ 15.0, 110.8, 112.5, 123.6, 124.6, 126.2, 127.7,
127.9, 129.2, 131.7, 136.5, 152.7, 155.2. Anal. (C16H13
ClJ N2O3S2) C, H, N.
-
N-(4-Ch lor op h en yl)-N′-(ben zo[2,1,3]th ia d ia zol-4-ylsu l-
fon yl)-S-m eth ylp seu d oth iou r ea (8g): MS m/z 339 (M+
+
1); 1H NMR (300 MHz, DMSO-d6) δ 2.42 (s, 3H, SCH3), 7.38-
7.45 (m, 4H, ArH), 7.86 (dd, J ) 8.8 Hz, 7.1 Hz, 1H, ArH),
8.26 (d, J ) 7.1 Hz, 1H, ArH), 8.37 (d, J ) 8.8 Hz, 1H, ArH),
9.85 (s, 1H, NH); 13C NMR (75 MHz, DMSO-d6) δ 14.9, 125.8,
127.0, 128.7, 129.6, 130.9, 133.4, 136.1, 148.8, 155.0, 166.8.
Anal. (C14H11ClN4O2S3) C, H, N.
N-(4-Ch lor op h en yl)-N′-[(2-p yr id -2-ylt h iop h en e-5-yl)-
su lfon yl]-S-m eth ylp seu d oth iou r ea (8h ): MS m/z 424 (M+);
1H NMR (300 MHz, DMSO-d6) δ 2.54 (s, 3H, SCH3), 7.36-
7.49 (m, 5H, ArH), 7.68 (d, J ) 4.0 Hz, 1H, thiophene-H), 7.81
(d, J ) 4.0 Hz, 1H, thiophene-H), 7.87-7.92 (m, 1H, ArH), 8.04
(d, J ) 7.8 Hz, 1H, ArH), 8.58 (d, J ) 4.8 Hz, 1H, ArH), 9.80
(s, 1H, NH); 13C NMR (75 MHz, DMSO-d6) δ 15.3, 119.7, 124.1,
124.9, 127.7, 129.2, 131.5, 132.4, 136.6, 137.9, 144.5, 150.0,
150.9. Anal. (C17H14ClN3O2S3) C, H, N.
N-(3,4-Dich lor oph en yl)-N′-[(5-ch lor o-3-m eth ylben zo[b]-
1
th iop h en -2-yl)su lfon yl]th iou r ea (7r ): MS m/z 46 (M+); H
NMR (300 MHz, DMSO-d6) δ 2.48 (s, 3H, CH3), 7.40-7.45 (m,
2H, Ar-H), 7.66 (dd, J ) 9.0 Hz, J ) 2.3 Hz, 1H, Ar-H), 7.84
(d, J ) 2.0 Hz, 1H, Ar-H), 7.95 (d, J ) 8.6 Hz, 1H, Ar-H), 8.22
(d, J ) 2.3 Hz, 1H, Ar-H), 9.52 (br s, 1H, Ar-H); 13C NMR (75
MHz, DMSO-d6) δ 12.3, 120.0, 121.2, 122.6, 122.7, 124.5, 125.9,
129.7, 130.2, 130.6, 131.3, 137.4, 141.4, 141.6, 183.3. Anal.
(C16H10Cl3N2O2S3Na‚H2O) C, H, N.
N-(4-Ch lor op h en yl)-N′-[(5-isoxa zol-3-ylt h iop h en e-2-
1
yl)su lfon yl]-S-m eth ylp seu d oth iou r ea (8i): H NMR (300
P r ep a r a t ion of N,N′-Disu b st it u t ed S-m et h ylp seu d o-
th iou r ea s 8a -r : Gen er a l P r oced u r e. Sodium hydroxide
solution (1 N, 5.0 mL) was added to a solution containing the
appropriate sulfonamide 6a -p (5.0 mmol) in acetone (100 mL).
A solution of the appropriate isothiocyanates (5.0 mmol) in
acetone (25 mL) was added. After the mixture was stirred at
room temperature for 4 h, methyl iodide (5.6 mmol) was added
to the filtrate. The reaction mixture was stirred for 30 min
before being neutralized with 1 N acetic acid (5.5 mL). The
solid was collected and recrystallized from the appropriate
solvent. The mp and yield data of the compounds are sum-
marized in Table 3. The analytical data are given below.
N-[(4-Met h ylp h en yl)su lfon yl]-N′-(4-ch lor op h en yl)-S-
m eth ylp seu d oth iou r ea (8a ): MS m/z 354 (M+); 1H NMR (300
MHz, DMSO-d6) δ 2.38 (s, 3H, CH3), 2.47 (s, 3H, SCH3), 7.35-
7.38 (m, 4H, ArH), 7.45 (d, J ) 8.4 Hz, 2H, ArH), 7.74 (d, J )
8.0 Hz, 2H, ArH), 9.66 (s, 1H, NH); 13C NMR (75 MHz, DMSO-
d6) δ 15.2, 21.3, 126.3, 127.6, 129.1, 129.7, 131.3, 136.7, 139.8,
142.8, 166.4. Anal. (C15H15ClN2O2S2) C, H, N.
MHz, DMSO-d6) δ 2.56 (s, 3H, SCH3), 7.09 (d, J ) 2.0 Hz, 1H,
ArH), 7.40 (d, J ) 8.8 Hz, 2H, ArH), 7.49 (d, J ) 8.8 Hz, 2H,
ArH), 7.71 (d, J ) 4.0 Hz, 1H, ArH), 7.75 (d, J ) 4.0 Hz, 1H,
ArH), 8.74 (d, J ) 2.0 Hz, 1H, ArH), 9.89 (s, 1H, NH); 13C NMR
(75 MHz, DMSO-d6) δ 15.0, 101.4, 127.3, 127.4, 128.8, 131.7,
132.5, 136.0, 145.3, 151.9, 162.0, 167.3; HREIMS (exact mass
HREMS) calcd for C15H12ClN3O3S3 m/e 412.9729, found
412.9729.
N-(4-Ch lor op h en yl)-N′-[[5-(p h en ylsu lfon yl)th iop h en e-
2-yl]su lfon yl]-S-m eth ylp seu d oth iou r ea (8j): MS m/z 440
(M+ - 47); 1H NMR (300 MHz, DMSO-d6) δ 2.56 (s, 3H, SCH3),
7.56-7.49 (m, 4H, ArH), 7.64-7.87 (m, 5H, ArH), 8.02-8.07
(m, 2H, ArH), 9.99 (brs, 1H, NH); 13C NMR (100 MHz, DMSO-
d6) δ 15.4, 106.2, 118.9, 127.7, 127.8, 127.8, 129.2, 130.5, 131.2,
131.3, 134.1, 134.6, 134.9, 205.4. Anal. (C18H15ClN2O4S4) C,
H, N.
N-(4-Ch lor op h en yl)-N′-[[4-(p h en ylsu lfon yl)th iop h en e-
2-yl]su lfon yl]-S-m eth ylp seu d oth iou r ea (8k ): MS m/z 438
(M+ - 49); 1H NMR (300 MHz, DMSO-d6) δ 3.17 (s, 3H, SCH3),

Sulfonyl-N-hydroxyguanidine Derivatives as Antitumor Agents
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 14 2283
7.29 (d, J ) 8.7 Hz, 2H, ArH), 7.45 (d, J ) 8.7 Hz, 2H, ArH),
7.64-7.78 (m, 3H, ArH), 7.93 (s, 1H, ArH), 8.03 (d, J ) 7.4
Hz, 2H, ArH), 8.72 (d, J ) 1.6 Hz, 1H, ArH), 9.92 (s, 1H, NH);
13C NMR (75 MHz, DMSO-d6) δ 15.4, 127.8, 127.9, 128.3, 129.2,
130.3, 131.8, 134.5, 136.3, 137.8, 140.8, 141.1, 147.3, 167.9.
Anal. (C18H15ClN2O4S4‚1/2H2O) C, H, N.
N-(4-Ch lor op h en yl)-N′-[[4-(2-ch lor o-2-cya n op h en oxy)-
p h en yl]su lfon yl]-S-m et h ylp seu d ot h iou r ea (8l): MS m/z
491 (M+ - 1); 1H NMR (300 MHz, DMSO-d6) δ 2.50 (s, 3H,
SCH3), 7.16 (d, J ) 9.2 Hz, 1H, ArH), 7.32-7.47 (m, 6H, ArH),
7.57 (d, J ) 8.4 Hz, 1H, ArH), 7.73 (t, J ) 8.4 Hz, 1H, ArH),
7.94 (d, J ) 8.8 Hz, 2H, ArH), 9.74 (s, 1H, NH); 13C NMR (75
MHz, DMSO-d6) δ 15.0, 104.9, 113.0, 117.8, 119.1, 125.4, 127.3,
128.8, 129.0, 131.0, 136.1, 136.3, 136.7, 138.6, 157.7, 158.8,
166.3. Anal. (C21H15Cl2N3O3S2) C, H, N.
N-(4-Ch lor op h en yl)-N′-[[4-(2-ch lor o-6-n itr op h en oxy)-
p h en yl]su lfon yl]-S-m eth ylp seu d oth iou r ea (8m ): MS m/z
512 (M+); 1H NMR (300 MHz, DMSO-d6) δ 2.49 (s, 3H, SCH3),
8.07 (d, J ) 8.9 Hz, 2H, ArH), 7.35 (d, J ) 8.9 Hz, 2H, ArH),
7.44 (d, J ) 8.9 Hz, 2H, ArH), 7.64 (t, J ) 8.2 Hz, 1H, ArH),
7.86 (d, J ) 8.9 Hz, 2H, ArH), 8.09 (dd, J ) 8.2 Hz, J ) 1.5
Hz, 1H, ArH), 8.19 (dd, J ) 8.2 Hz, J ) 1.6 Hz, 1H, ArH),
9.71 (s, 1H, NH); 13C NMR (75 MHz, DMSO-d6) δ 15.2, 115.7,
125.5, 127.6, 128.3, 129.1, 129.2, 129.5, 131.3, 136.4, 136.7,
137.3, 142.6, 144.6, 159.3, 166.6. Anal. (C20H15Cl2N3O5S2) C,
H, N.
N-(4-Ch lor op h en yl)-N′-[[3,5-d ich lor o-4-(4-n itr op h en ox-
y)p h en yl]su lfon yl]-S-m eth ylp seu d oth iou r ea (8n ): MS m/z
547 (M+); 1H NMR (300 MHz, DMSO-d6) δ 2.56 (s, 3H, SCH3),
7.18 (d, J ) 9.2 Hz, 2H, ArH), 7.41 (d, J ) 8.8 Hz, 2H, ArH),
7.49 (d, J ) 8.8 Hz, 2H, ArH), 8.10 (s, 2H, ArH), 8.26 (d, J )
9.2 Hz, 2H, ArH), 9.96 (s, 1H, NH); 13C NMR (75 MHz, DMSO-
d6) δ 15.4, 116.2, 126.8, 128.1, 129.2, 129.5, 130.3, 131.8, 136.5,
142.3, 143.4, 148.2, 160.6, 167.8. Anal. (C20H14Cl3N3S2) C, H,
N.
N-(4-Ch lor op h en yl)-N′-[[4-(2-ch lor o-4-n itr op h en oxy)-
3,5-d ich lor op h en yl]su lfon yl]-S-m et h ylp seu d ot h iou r ea
(8o): MS m/z 533 (M+ - 48); 1H NMR (300 MHz, DMSO-d6) δ
2.56 (s, 3H, SCH3), 7.01 (d, J ) 9.1 Hz, 1H, ArH), 7.41 (d, J )
8.8 Hz, 2H, ArH), 7.49 (d, J ) 8.8 Hz, 2H, ArH), 8.10-8.14
(m, 3H, ArH), 8.54 (d, J ) 2.7 Hz, 1H, ArH), 9.96 (s, 1H, NH);
13C NMR (75 MHz, DMSO-d6) δ 15.4, 115.4, 122.4, 125.0, 126.9,
128.1, 128.2, 129.2, 131.7, 136.7, 142.7, 143.6, 148.0, 156.0.
Anal. (C20H13Cl4N3O5S2) C, H, N.
N-(4-Ch lor op h en yl)-N′-[(4-n -bu toxyp h en yl)su lfon yl]-S-
m eth ylp seu d oth iou r ea (8p ): MS m/z 412 (M+ - 1); 1H NMR
(300 MHz, DMSO-d6) δ 0.93 (t, J ) 7.3 Hz, 3H, CH3), 1.32-
1.43 (m, 2H, CH2), 1.67-1.76 (m, 2H, CH2), 2.46 (s, 3H, SCH3),
4.05 (t, J ) 6.4 Hz, 2H, OCH2), 7.07 (d, J ) 8.9 Hz, 2H, ArH),
7.36 (d, J ) 8.8 Hz, 2H, ArH), 7.45 (d, J ) 8.8 Hz, 2H, ArH),
7.78 (d, J ) 8.9 Hz, 2H, ArH), 9.63 (s, 1H, NH); 13C NMR (75
MHz, DMSO-d6) δ 14.0, 15.2, 19.1, 30.9, 68.0, 114.8, 127.6,
ethylamine (1.2 mL, 8.4 mmol) in chloroform (50 mL). The
solution was refluxed for 48 h, and the solvent was removed
by evaporation to produce a solid residue. Ether (20 mL) was
added to the residue, and the white precipitate was collected
by filtration. The solid was then heated with toluene, and the
undissolved solid was removed by filtration. The filtrate was
again evaporated to obtain a solid which was subsequently
recrystallized from the appropriate solvent to yield the desired
compound.
N-[(4-Meth ylp h en yl)su lfon yl]-N′-(4-ch lor op h en yl)-N′′-
h yd r oxygu a n id in e (4a ): MS m/z 294 (M+
- 32); 1H NMR
(300 MHz, DMSO-d6) δ 2.36 (s, 3H, CH3), 7.31-7.36 (m, 4H,
ArH), 7.45 (d, J ) 8.8 Hz, 2H, ArH), 7.71 (d, J ) 8.1 Hz, 2H,
ArH), 9.40 (s, 1H, NH), 9.69 (brs, 2H, NH and OH); 13C NMR
(100 MHz, DMSO-d6) δ 20.9, 124.7, 125.9, 128.2, 128.3, 129.1,
136.2, 140.6, 141.6, 154.0. Anal. (C14H14ClN3O3S) C, H, N.
N-(P h en ylsu lfon yl)-N′-(4-ch lor op h en yl)-N′′-h yd r oxy-
1
gu a n id in e (4b): MS m/z 328 (M+ + 2); H NMR (300 MHz,
DMSO-d6) δ 7.34 (d, J ) 8.9 Hz, 2H, ArH), 7.44 (d, J ) 8.9
Hz, 2H, ArH), 7.49-7.61 (m, 3H, ArH), 7.81-7.84 (dd, J )
7.8 Hz, J ) 1.8 Hz, 2H, ArH), 9.42 (s, 1H, NH), 9.81 (brs, 2H,
NHOH); 13C NMR (100 MHz, DMSO-d6) δ 124.8, 125.8, 128.2,
128.4, 128.7, 131.6, 136.2, 143.4, 154.1. Anal. (C13H12
ClN3O3S) C, H, N.
-
N-(4-Ch lor oph en yl)-N′-[[3,5-bis(tr iflu or om eth yl)ph en yl]-
su lfon yl]-N′′-h yd r oxygu a n id in e (4c). After the reaction
was complete, the solvent was removed in vacuo, and the
residue was purified by column chromatography (silica gel,
solvent system: n-hexane/ethyl acetate ) 1:1). The Rf ) 0.44
fraction was collected to obtain 4c (318 mg, 21.93%): MS m/z
461.4 (M+); 1H NMR (300 MHz, DMSO-d6) δ 7.30-7.40 (m,
4H, ArH), 8.32 (s, 2H, ArH), 8.37 (s, 1H, ArH), 9.61 (s, 1H,
NH), 10.01 (s, 1H, NH), 10.27 (s, 1H, OH); 13C NMR (75 MHz,
DMSO-d6) δ 123.1 (q, J ) 272 Hz, CF3), 125.7, 126.0, 126.9,
128.6, 129.4, 131.3 (q, J ) 33 Hz, CCF3), 136.3, 146.8, 154.2.
Anal. (C15H10F6ClN3O3S) C, H, N.
N-(4-Ch lor op h en yl)-N′-[(5-ch lor o-3-m et h ylb en zo[b]-
th iop h en e-2-yl)su lfon yl]-N′′-h yd r oxygu a n id in e (4d ). Af-
ter the reaction was complete, the precipitate was removed,
and the filtrate, after concentration in vacuo to dryness, was
purified by column chromatography (silica gel, solvent sys-
tem: chloroform) to obtain two products. The Rf ) 0.28
fraction was collected to give 4d (0.62 g, 40.9%): mp 245-247
°C dec; MS m/z 413 (M+ - 17); 1H NMR (300 MHz, DMSO-d6)
δ 2.52 (s, 3H, CH3), 7.35 (d, J ) 8.9 Hz, 2H, ArH), 7.45 (d, J
) 8.9 Hz, 2H, ArH), 7.52 (dd, J ) 8.7 Hz, J ) 1.9 Hz, 1H,
ArH), 7.96 (d, J ) 1.9 Hz, 1H, ArH), 8.05 (d, J ) 8.7 Hz, 1H,
ArH), 9.60 (s, 1H, NH), 9.97 (s, 2H, NH and OH); 13C NMR
(75 MHz, DMSO-d6) δ 12.3, 123.3, 124.9, 125.6, 127.1, 128.6,
129.2, 130.5, 133.4, 136.7, 141.4, 154.5; HREIMS (exact mass
HREMS) calcd for C16H13O3S2N3Cl2 m/z 428.9775, found
428.9774. The Rf ) 0.37 fraction was collected and recrystal-
lized from ethanol and acetonitrile (v/v ) 1:1) to furnish 9d
(203 mg, 15.5%): mp 256-257 °C dec; MS m/z 413 (M+ - 1);
1H NMR (300 MHz, DMSO-d6) δ 2.58 (s, 3H, CH3), 7.12 (s,
2H, NH2), 7.38 (s, 4H, ArH), 7.53 (dd, J ) 8.7 Hz, J ) 2.1 Hz,
1H, ArH), 7.97 (d, J ) 2.1 Hz, 1H, ArH), 8.05 (d, J ) 8.7 Hz,
1H, ArH), 9.34 (s, 1H, NH); 13C NMR (75 MHz, DMSO-d6) δ
12.3, 123.4, 123.9, 125.0, 127.2, 128.6, 129.1, 130.6, 133.6,
136.7, 136.8, 141.4, 141.8, 155.0. Anal. (C16H13O2S2N3Cl2) C,
H, N.
128.8, 129.1, 131.2, 134.2, 136.8, 161.8, 166.1. Anal. (C18H21
-
ClN2O3S2‚1/4H2O) C, H, N.
N-(3,4-Dich lor op h en yl)-N′-(4-tolylsu lfon yl)-S-m eth yl-
p seu d oth iou r ea (8q): MS m/z 388 (M+); 1H NMR (300 MHz,
DMSO-d6) δ 2.38 (s, 3H, SCH3), 2.54 (s, 3H, CH3), 7.36-7.41
(m, 3H, ArH), 7.64 (d, J ) 8.6 Hz, 1H, ArH), 7.68 (d, J ) 2.3
Hz, 1H, ArH), 7.73 (d, J ) 8.3 Hz, 2H, ArH), 9.66 (s, 1H, NH);
13C NMR (75 MHz, DMSO-d6) δ 15.3, 21.4, 125.2, 126.6, 126.7,
129.3, 129.8, 130.9, 131.2, 138.0, 139.7. Anal. (C15H14
Cl2N2O2S2) C, H, N.
-
N -(4-Ch lor op h e n yl)-N ′-(b e n zo[b]t h iop h e n e -2-ylsu l-
fon yl)-N′′-h yd r oxygu a n id in e (4e). After the reaction was
complete, the filtrate was concentrated in vacuo to dryness
and purified by column chromatography (silica gel, solvent
system: chloroform/methanol ) 98:2) to obtain two products.
The Rf ) 0.16 fraction was collected to give 4e (0.28 g, 27%):
mp 264-266 °C; MS m/z 381 (M+); 1H NMR (300 MHz, DMSO-
d6) δ 7.36-7.39 (m, 2H, ArH), 7.45-7.50 (m, 4H, ArH), 7.96-
8.05 (m, 2H, ArH), 9.57 (s, 1H, NH), 9.99 (s, 2H, OH and NH);
13C NMR (75 MHz, DMSO-d6) δ 123.3, 123.8, 125.6, 125.7,
125.8, 127.1, 128.6, 129.1, 136.4, 137.9, 140.8, 145.2, 154.4.
Anal. (C15H12ClN3O3S2) C, H, N. The Rf ) 0.27 fraction was
collected and recrystallized from methanol to furnish 9e (25
N-(3,4-Dich lor oph en yl)-N′-[(5-ch lor o-3-m eth ylben zo[b]-
t h iop h en e-2-yl)su lfon yl]-S-m et h ylp seu d ot h iou r ea (8r ):
1
MS m/z 478 (M+ - 1); H NMR (300 MHz, DMSO-d6) δ 2.58
(s, 3H, SCH3), 2.61 (s, 3H, SCH3), 7.43 (dd, J ) 8.7 Hz, J )
2.4 Hz, 1H, ArH), 7.56 (dd, J ) 8.6 Hz, J ) 2.0 Hz, 1H, ArH),
7.68 (d, J ) 8.7 Hz, 1H, ArH), 8.07 (d, J ) 8.6 Hz, 1H, ArH),
9.86 (s, 1H, NH); 13C NMR (75 MHz, DMSO-d6) δ 12.4, 15.5,
96.6, 123.6, 125.0, 125.4, 127.0, 127.6, 127.7, 130.7, 131.0,
131.3, 134.9, 137.7, 141.1, 201.5, 209.8. Anal. (C17H13
Cl3N2O2S3) C, H, N.
-
P r ep a r a t ion of N,N′-Disu b st it u t ed N′′-H yd r oxy-
gu a n id in es 4a -r : Gen er a l P r oced u r e. The appropriate
S-methyl pseudourea 8a -r (2.8 mmol) was added to a stirred
solution of hydroxylamine hydrochloride (7.4 mmol) and tri-
1
mg, 2.6%): mp 205-206 °C; MS m/z 367 (M+); H NMR (300
MHz, DMSO-d6) δ 7.15 (s, 2H, NH2), 7.37 (s, 4H, ArH), 7.46-

2284 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 14
Chern et al.
7.50 (m, 2H, ArH), 7.95 (s, 1H, ArH), 7.97-8.06 (m, 2H, ArH),
9.33 (s, 1H, NH). Anal. (C15H12ClN3O2S2) C, H, N.
129.2, 130.3, 130.5, 134.9, 136.5, 140.9, 145.4, 155.2. Anal.
(C17H14ClN3O4S3) C, H, N.
N-(4-Ch lor op h en yl)-N′-(b en zofu r a n -2-ylsu lfon yl)-N′′-
h yd r oxygu a n id in e (4f): MS m/z 366 (M+); 1H NMR (300
MHz, DMSO-d6) δ 7.33-7.50 (m, 7H, ArH), 7.69 (d, J ) 8.2
Hz, 1H, ArH), 7.77 (d, J ) 7.7 Hz, 1H, ArH), 9.62 (s, 1H, NH),
10.09 (s, 2H, NH and OH); 13C NMR (75 MHz, DMSO-d6) δ
106.6, 109.4, 112.3, 123.3, 124.4, 125.6, 126.5, 127.4, 128.6,
129.1, 136.4, 154.0, 154.2. Anal. (C15H12ClN3O4S) C, H, N.
N-(4-Ch lor op h en yl)-N′-(ben zo[2,1,3]th ia d ia zol-4-ylsu l-
N-(4-Ch lor op h en yl)-N′-[[4-(p h en ylsu lfon yl)th iop h en e-
2-yl]su lfon yl]-N′′-h yd r oxygu a n id in e (4k ). Compound 4k
(Rf ) 0.4) was separated in 19.8% yield by column chroma-
tography (silica gel, solvent system: chloroform): MS m/z 413
(M+ - 59); 1H NMR (300 MHz, DMSO-d6) δ 7.34 (s, 4H, ArH),
7.64-7.75 (m, 3H, ArH), 7.81 (d, J ) 1.6 Hz, 1H, thiophene-
H), 8.01 (d, J ) 7.4 Hz, 2H, ArH), 8.63 (d, J ) 1.6 Hz, 1H,
thiophene-H), 9.59 (s, 1H, NH), 10.04 (s, 1H, OH), 10.15 (s,
1H, NH); 13C NMR (75 MHz, DMSO-d6) δ 106.2, 125.8, 127.0,
127.7, 128.6, 129.3, 130.3, 134.5, 136.2, 136.8, 140.8, 140.9,
154.1. Anal. (C17H14ClN3O5S3) C, H, N. The Rf ) 0.47 fraction
was collected and recrystallized from chloroform to give 9k
fon yl)-N′′-h yd r oxygu a n id in e (4g). Compound 4g (Rf
)
0.04) was separated in 31.2% yield by column chromatography
(silica gel, solvent system: chloroform/methanol ) 99:1): MS
m/z 366.5 (M+ - 17); 1H NMR (300 MHz, DMSO-d6) δ 7.24 (d,
J ) 8.9 Hz, 2H, ArH), 7.43 (d, J ) 8.9 Hz, 2H, ArH), 7.83 (dd,
J ) 8.8 Hz, 7.1 Hz, 1H, ArH), 8.22 (dd, J ) 7.1 Hz, 1.1 Hz,
1H, ArH), 8.31 (dd, J ) 8.8 Hz, 1.1 Hz, 1H, ArH), 9.47 (s, 1H,
NH), 9.95 (s, 2H, NH and OH); 13C NMR (75 MHz, DMSO-d6)
δ 125.0, 125.5, 128.4, 128.6, 129.2, 129.4, 135.1, 136.5, 154.6,
155.5, 158.3. Anal. (C13H10ClN5O3S2) C, H, N. The Rf ) 0.05
fraction was collected and recrystallized from acetonitrile to
furnish 9g (104 mg, 8.9%): mp 260 °C dec; MS m/z 368.5 (M+
- 19); 1H NMR (300 MHz, DMSO-d6) δ 7.17 (s, 2H, NH2),
7.28-7.37 (AB q, J ) 9.0 Hz, 4H, ArH), 7.84 (dd, J ) 8.8 Hz,
7.1 Hz, 1H, ArH), 8.22 (dd, J ) 7.1 Hz, 1.1 Hz, 1H, ArH), 8.33
(dd, J ) 8.8 Hz, 1.1 Hz, 1H, ArH), 9.24 (s, 1H, NH); 13C NMR
(75 MHz, DMSO-d6) δ 116.3, 123.3, 125.6, 128.0, 129.0, 129.2,
135.1, 137.1, 155.1, 155.5, 170.5. Anal. (C13H10ClN5O2S2) C,
H, N.
1
(65 mg, 0.06%): MS m/z 438 (M+ - 18); H NMR (300 MHz,
DMSO-d6) δ 7.19 (s, 2H, NH2), 7.27 (d, J ) 8.9 Hz, 2H, ArH),
7.36 (d, J ) 8.9 Hz, 2H, ArH), 7.66 (t, J ) 7.4 Hz, 2H, ArH),
7.75 (t, J ) 7.4 Hz, 1H, ArH), 7.86 (d, J ) 1.6 Hz, 1H,
thiophene-H), 8.02 (d, J ) 7.4 Hz, 2H, ArH), 8.65 (d, J ) 1.6
Hz, 1H, thiophene-H), 9.30 (s, 1H, NH); 13C NMR (75 MHz,
DMSO-d6) δ 124.0, 124.2, 127.2, 127.8, 128.8, 129.1, 129.2,
130.3, 134.5, 136.6, 137.0, 155.1. Anal. (C17H14ClN3O4S3) C,
H, N.
N-(4-Ch lor op h en yl)-N′-[[4-(3-ch lor o-2-cya n op h en oxy)-
p h en yl]su lfon yl]-N′′-h yd r oxygu a n id in e (4l). Compound
4l (Rf ) 0.23) was separated in 44.1% yield by column
chromatography (silica gel, solvent system: chloroform:metha-
nol ) 98:2): MS m/z 418 (M+ - 59); 1H NMR (300 MHz,
DMSO-d6) δ 8.11 (d, J ) 8.3 Hz, 1H, ArH), 7.30-7.37 (m, 4H,
ArH), 7.45 (d, J ) 8.9 Hz, 2H, ArH), 7.56 (d, J ) 8.2 Hz, 1H,
ArH), 7.72 (t, J ) 8.3 Hz, 1H, ArH), 7.90 (d, J ) 8.7 Hz, 2H,
ArH), 9.47 (s, 1H, NH), 9.88 (s, 2H, NH and OH); 13C NMR
(75 MHz, DMSO-d6) δ 104.8, 113.0, 117.5, 119.0, 124.8, 125.2,
128.2, 128.4, 128.5, 136.0, 136.2, 136.5, 140.0, 154.0, 157.1,
159.0. Anal. (C20H14Cl2N4O4S) C, H, N.
N-(4-Ch lor op h en yl)-N′-[(2-p yr id -2-ylt h iop h en e-5-yl)-
su lfon yl]-N′′-h yd r oxygu a n id in e (4h ). Compound 4h (Rf )
0.29) was separated in 52.2% yield by column chromatography
(silica gel, solvent system: chloroform/methanol ) 98:2): MS
m/z 411 (M+ + 1); 1H NMR (300 MHz, DMSO-d6) δ 7.33-7.39
(m, 3H, ArH), 7.47 (d, J ) 8.9 Hz, 2H, ArH), 7.61 (d, J ) 4.0
Hz, 1H, thiophene-H), 7.77 (d, J ) 4.0 Hz, 1H, thiophene-H),
7.88 (td, J ) 7.8 Hz, J ) 1.6 Hz, 1H, ArH), 8.00 (d, J ) 7.8
N-(4-Ch lor op h en yl)-N′-[[4-(2-ch lor o-6-n itr op h en oxy)-
p h en yl]su lfon yl]-N′′-h yd r oxygu a n id in e (4m ). Compound
4m (Rf ) 0.36) was separated in 35.2% yield by column
chromatography (silica gel, solvent system: chloroform:metha-
Hz, 1H, ArH), 9.55 (s, 1H, NH), 9.97 (s, 2H, NH and OH); 13
C
NMR (75 MHz, DMSO-d6) δ 119.6, 123.9, 124.7, 125.4, 128.6,
129.0, 131.3, 136.5, 137.8, 146.1, 148.9, 150.0, 151.1, 154.3.
Anal. (C16H13ClN4O3S2) C, H, N. The Rf ) 0.35 fraction was
collected and recrystallized from methanol to furnish 9h (100
1
nol ) 98:2): MS m/z 364.5 (M+ - 132); H NMR (300 MHz,
DMSO-d6) δ 7.03 (d, J ) 8.8 Hz, 2H, ArH), 7.33 (d, J ) 8.9
Hz, 2H, ArH), 7.43 (d, J ) 8.9 Hz, 2H, ArH), 7.63 (t, J ) 8.2
Hz, 1H, ArH), 7.81 (d, J ) 8.8 Hz, 2H, ArH), 8.08 (dd, J ) 8.2
Hz, J ) 1.5 Hz, 1H, ArH), 8.18 (dd, J ) 8.2 Hz, J ) 1.5 Hz,
1H, ArH), 9.46 (s, 1H, NH), 9.85 (brs, 2H, NH and OH); 13C
NMR (75 MHz, DMSO-d6) δ 115.5, 125.2, 125.4, 128.3, 128.6,
128.7, 128.8, 129.6, 136.4, 136.6, 138.3, 142.7, 144.7, 154.3,
158.9. Anal. (C19H14Cl2N4O6S) C, H, N.
1
mg, 6.5%): mp 229-230 °C; MS m/z 394 (M+); H NMR (300
MHz, DMSO-d6) δ 7.13 (s, 2H, NH2), 7.33-7.41 (m, 5H, ArH),
7.60 (d, J ) 4.0 Hz, 1H, thiophene-H), 8.77 (d, J ) 4.0 Hz, 1H,
thiophene-H), 8.88 (td, J ) 7.6 Hz, J ) 1.7 Hz, 1H, ArH), 8.00
(d, J ) 7.6 Hz, 1H, ArH), 8.55 (dd, J ) 5.0 Hz, J ) 1.1 Hz, 1H,
ArH), 9.30 (s, 1H, NH); 13C NMR (75 MHz, DMSO-d6) δ 119.2,
123.2, 123.4, 124.4, 127.0, 128.7, 130.6, 136.5, 137.4, 145.6,
148.6, 149.5, 150.6, 154.6. Anal. (C16H13ClN4O2S2) C, H, N.
N-(4-Ch lor op h en yl)-N′-[(5-isoxa zol-3-ylt h iop h en e-5-
yl)su lfon yl]-N′′-h yd r oxygu a n id in e (4i). Compound 4i (Rf
) 0.19) was separated in 37.5% yield by column chromatog-
raphy (silica gel, solvent system: chloroform/methanol ) 98:
2): MS m/z 396 (M+ - 3); 1H NMR (300 MHz, DMSO-d6) δ
7.05 (d, J ) 1.8 Hz, 1H, thiophene-H), 7.41 (q, J ) 9.0 Hz, 4H,
ArH), 7.68 (s, 2H, ArH), 7.72 (d, J ) 1.8 Hz, thiophene-H),
N-(4-Ch lor op h en yl)-N′-[[3,5-d ich lor o-4-(4-n it r op h en -
oxy)p h en yl]su lfon yl]-N′′-h yd r oxygu a n id in e (4n ). Com-
pound 4n (Rf ) 0.59) was separated in 23.9% yield by column
chromatography (silica gel, solvent system: ethyl acetate:
1
hexane ) 1:1): MS m/z 473.8 (M+ - 58); H NMR (300 MHz,
DMSO-d6) δ 7.16 (d, J ) 9.2 Hz, 2H, ArH), 7.38 (d, J ) 8.9
Hz, 2H, ArH), 7.46 (d, J ) 8.9 Hz, 2H, ArH), 8.06 (s, 2H, ArH),
8.26 (d, J ) 9.2 Hz, 2H, ArH), 9.61 (s, 1H, NH), 10.06 (s, 1H,
OH), 10.16 (s, 1H, NH); 13C NMR (75 MHz, DMSO-d6) δ 115.7,
123.6, 125.4, 126.4, 127.4, 128.3, 128.8, 136.0, 143.0, 143.3,
147.3, 153.8, 160.3. Anal. (C19H13Cl3N4O6S) C, H, N.
9.60 (s, 1H, NH), 10.05 (s, 2H, NH and OH). Anal. (C14H11
-
ClN4O4S2) C, H, N.
N-(4-Ch lor op h en yl)-N′-[[5-(p h en ylsu lfon yl)th iop h en e-
2-yl]su lfon yl]-N′′-h yd r oxygu a n id in e (4j). Compound 4j (Rf
) 0.35) was separated in 11.6% yield by column chromatog-
raphy (silica gel, solvent system: chloroform): MS m/z 413
(M+ - 59); 1H NMR (300 MHz, DMSO-d6) δ 7.32 (s, 4H, ArH),
7.59 (d, J ) 4.0 Hz, 1H, thiophene-H), 7.65-7.78 (m, 3H, ArH),
8.03 (d, J ) 7.1 Hz, 2H, ArH), 9.65 (s, 1H, NH), 10.05 (s, 1H,
OH), 10.20 (s, 1H, NH); 13C NMR (75 MHz, DMSO-d6) δ 125.6,
125.9, 127.6, 128.6, 129.4, 130.2, 130.4, 134.0, 134.8, 134.8,
136.1, 141.0, 145.2, 153.4, 154.2. Anal. (C17H14ClN3O5S3) C,
H, N. The Rf ) 0.44 fraction was collected and recrystallized
from toluene to give 9j (37 mg, 2.7%): mp 233-234 °C; MS
m/z 349 (M+ - 107); 1H NMR (300 MHz, DMSO-d6) δ 7.20 (s,
2H, NH2), 7.28 (d, J ) 8.8 Hz, 2H, ArH), 7.37 (d, J ) 8.8 Hz,
2H, ArH), 7.61-7.83 (m, 4H, ArH), 7.84 (d, J ) 3.7 Hz, 1H,
thiophene-H), 8.03 (d, J ) 7.5 Hz, 2H, ArH), 9.36 (s, 1H, NH);
13C NMR (75 MHz, DMSO-d6) δ 106.2, 106.6, 124.3, 127.6,
N-(4-Ch lor op h en yl)-N′-[[3,5-d ich lor o-4-(2-ch lor o-4-n i-
tr op h en oxy)p h en yl]su lfon yl]-N′′-h yd r oxygu a n id in e (4o).
Compound 4o (Rf ) 0.53) was separated in 30.6% (266 mg)
yield by column chromatography (silica gel, solvent system:
1
ethyl acetate:hexane ) 1:1): MS m/z 552 (M+ + 4); H NMR
(300 MHz, DMSO-d6) δ 6.96 (d, J ) 9.2 Hz, 2H, ArH), 7.37 (d,
J ) 8.9 Hz, 2H, ArH), 7.46 (d, J ) 8.9 Hz, 2H, ArH), 8.08 (s,
2H, ArH), 8.11 (dd, J ) 9.2 Hz, J ) 2.7 Hz, 1H, ArH), 8.53 (d,
J ) 2.7 Hz, 1H, ArH), 9.61 (s, 1H, NH), 10.08 (brs, 2H, NH
and OH); 13C NMR (75 MHz, DMSO-d6) δ 115.3, 122.4, 125.0,
125.8, 126.9, 127.9, 128.7, 128.9, 129.2, 136.4, 143.6, 144.0,
147.5, 154.1, 156.1. Anal. (C19H12Cl4O6S‚H2O) C, H, N. The
Rf ) 0.56 fraction was collected and recrystallized from
acetonitrile to give 9o (100 mg, 12.1%): mp 193 °C; MS m/z
550 (M+); 1H NMR (300 MHz, DMSO-d6) δ 6.99 (d, J ) 9.1
Hz, 1H, ArH), 7.23 (s, 2H, NH2), 7.34-7.41 (m, 4H, ArH),
8.08-8.12 (m, 3H, ArH), 8.53 (d, J ) 2.8 Hz, 1H, ArH), 9.31

Sulfonyl-N-hydroxyguanidine Derivatives as Antitumor Agents
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 14 2285
(s, 1H, NH); 13C NMR (75 MHz, DMSO-d6) δ 115.4, 122.4,
124.1, 125.0, 16.9, 127.7, 128.6, 129.1, 129.2, 136.8, 143.6,-
144.0, 147.6, 155.0, 156.0. Anal. (C19H12Cl4N4O5S) C, H, N.
N-(4-Ch lor op h en yl)-N′-[(4-n -b u t oxyp h en yl)su lfon yl]-
N′′-h yd r oxygu a n id in e (4p ). Compound 4p (Rf ) 0.51) was
separated in 34.3% (397 mg) yield by column chromatography
(silica gel, solvent system: ethyl acetate:hexane ) 3:7): MS
m/z 381 (M+ - 16); 1H NMR (300 MHz, DMSO-d6) δ 0.93 (t, J
) 7.3 Hz, 3H, CH3), 1.37-1.49 (m, 2H, CH2), 1.65-1.75 (m,
2H, CH2), 4.02 (t, J ) 6.4 Hz, 2H, OCH2), 7.03 (d, J ) 8.9 Hz,
2H, ArH), 7.35 (d, J ) 8.9 Hz, 2H, ArH), 7.47 (d, J ) 8.9 Hz,
2H, ArH), 7.74 (d, J ) 8.9 Hz, 2H, ArH), 9.38 (s, 1H, NH),
9.77 (s, 2H, NH and OH); 13C NMR (75 MHz, DMSO-d6) δ 14.0,
19.0, 31.0, 67.9, 114.6, 125.0, 128.4, 128.6, 135.6, 136.7, 154.3,
161.4, 162.5. Anal. (C17H20ClN3O4S) C, H, N.
N -(3,4-Dich lor op h e n yl)-N ′-(4-t olylsu lfon yl)-N ′′-h y-
d r oxygu a n id in e (4q). Compound 4q (Rf ) 0.36) was sepa-
rated in 24.9% (256 mg) yield by column chromatography
(silica gel, solvent system: methanol:chloroform ) 2:98): MS
m/z 375 (M+); 1H NMR (300 MHz, DMSO-d6) δ 2.36 (s, 3H,
CH3), 7.34 (d, J ) 8.1 Hz, 2H, ArH), 7.46-7.56 (m, 2H, ArH),
7.71-7.75 (m, 3H, ArH), 9.51 (s, 1H, NH), 9.92 (s, 2H, NH
and OH); 13C NMR (75 MHz, DMSO-d6) δ 20.9, 122.7, 124.0,
125.8, 129.2, 130.1, 130.4, 137.6, 140.5, 141.7, 153.4. Anal.
(C14H13Cl2N3O3S) C, H, N. The Rf ) 0.42 fraction was collected
to give 9q (230 mg, 23.9%): mp 190 °C; MS m/z 359 (M+ + 1);
1H NMR (300 MHz, DMSO-d6) δ 2.36 (s, 3H, CH3), 7.09 (s,
2H, NH2), 7.24 (dd, J ) 8.8 Hz, J ) 2.6 Hz, 1H, ArH), 7.35 (d,
J ) 8.2 Hz, 2H, ArH), 7.54 (d, J ) 8.8 Hz, 2H, ArH), 9.26 (s,
1H, NH); 13C NMR (75 MHz, DMSO-d6) δ 21.3, 106.6, 121.5,
122.8, 126.1, 129.7, 130.9, 138.5, 139.3, 142.3, 154.4, 160.9.
Anal. (C14H13Cl2N3O2S) C, H, N.
drogenase of viable cells.29-31 The viable cell number/well is
directly proportional to the production of formazan, which
following solubilization, can be measured spectrophotometri-
cally. Single-cell suspensions were obtained by mechanical
disaggregation of the floating cell line (MOLT-4) and by
trypsinization of the monolayer cultures (COLO 205, HepG2,
KB, and TSGH 8302) and counted by trypan blue exclusion.
The cells were then seeded into 96-well plates (Nunc 67008)
in a 180 µL volume using a multichannel pipet (Gilson) and
incubated for 24 h. The drug was dissolved in 10% DMSO
(Sigma, D-8779) and 90% DPBS solution. Drug solution (20
µL) was dispensed within appropriate wells (each treatment
group and control, N ) 3) at final drug concentrations from
100 µg/mL to 0.01 µg/mL by a 10-time dilution. Peripheral
wells for each plate (lacking cells) were utilized for drug blank
and medium/tetrazolium reagent blanks “background deter-
minations”. The cells were then incubated for another 72 h.
MTT (20 µL, 5 mg/mL) was added to each well and incubated
for a further 4 h. Culture plates containing MOLT-4 cells were
centrifuged at 1000 rpm for 5 min. Culture medium super-
natant (170 µL) was removed from each well and replaced with
200 µL/well DMSO using a multichannel pipet. Following
formazans solubilization, the absorbance of each well was
measured using an ELISA reader (Molecular Devices Emax)
at (545-690 nm) interfaced with IBM computer Softmax
software. Cell growth inhibition was calculated according to
(1 - (OD of drug treatment/OD of control)) × 100%. The IC50
values were obtained by determining the drug concentration
producing 50% growth inhibition by drawing the drug con-
centration vs growth inhibition percentage.
In Vivo An titu m or Tests. C3H/HeN mice (16-20 g, 5
weeks old) were obtained from the animal center of the Cheng-
Kung University and allowed to acclimate to their new
environment for one week. Murine K-1735/M2 melanoma cells
were obtained from Dr. Mien-Chie Hung (M.D. Anderson
Cancer Center, Houston, TX) and subcutaneously inoculated
in C3H/HeN mice. The tumors were maintained by serial
transplantation in C3H/HeN mice. Tumors were grown until
a size of approximately 15 × 20 mm. Tumors were then
resected, minced, and used for subcutaneous inoculation in
mice for the antitumor agent test. The tumors were grown
until approximately 150 mg (range 100-200 mg). The mice
were randomly divided into six mice per group. The com-
pounds (4o and LY181984) were resuspended in 2.5% cremo-
phor/saline. The desired dosage of compound was orally
administered for two cycles of 5 consecutive days [(qid × 5)2]
at day 5 and day 12. Tumor weight was estimated by two-
dimensional caliper measurements and calculated with the
formula for an ellipsoid. Tumor weight ) LW2/2, where L is
the major axis and W is the width of the tumor.2,32,33 The
percentage of tumor growth inhibition (TGI %) was calculated
as (1 mean tumor weight of treated group/mean tumor weight
of control group) × 100%. Moderate activity and significant
activity were defined as TGI of 58-89% and g90%, respec-
tively.32,34
N-(3,4-Dich lor oph en yl)-N′-[(5-ch lor o-3-m eth ylben zo[b]-
th iop h en e-2-yl)su lfon yl]-N′′-h yd r oxygu a n id in e (4r ): MS
m/z 466 (M+ + 1); 1H NMR (300 MHz, DMSO-d6) δ 2.56 (s,
3H, CH3), 7.45 (dd, J ) 8.8 Hz, J ) 2.4 Hz, 1H, ArH), 7.51-
7.57 (m, 2H, ArH), 7.73 (d, J ) 2.4 Hz, 1H, ArH), 7.97 (d, J )
2.0 Hz, 1H, ArH), 8.05 (d, J ) 8.6 Hz, 1H, ArH), 9.69 (s, 1H,
NH), 10.10 (brs, 2H, NH and OH); 13C NMR (75 MHz, DMSO-
d6) δ 12.3, 100.7, 106.6, 123.4, 123.5, 124.8, 124.9, 127.2, 130.5,
130.6, 133.4, 136.7, 137.7, 141.4, 153.8. Anal. (C16H12
Cl3N3O3S2) C, H, N.
-
X-r a y Cr ysta llogr a p h y. Crystals of 4g were grown from
a warm solution of the compound from methanol by slow
cooling. The X-ray diffraction data were collected for each
compound (to 2θ ) 120°) on a Rigaku AFC-5R (RU-300)
rotating anode X-ray diffractometer at 22 °C using the ω-2θ
scan mode with graphite-monochromated Cu KR radiation (λ
) 1.5418 Å). The power of the X-ray generator was set at 50
kV and 40 mA with 0.2 × 2 mm2 fine-focus anode cup. Unit
cell dimensions were determined using Cu KR1 radiation (λ
) 1.5406 Å). The crystallographic parameters are as follows:
space group P1h, a ) 10.573(1) Å, b ) 21.955(2) Å, c )
14.360(1) Å, â ) 110.65(1)°, no. of 3σ Fo’s ) 4585, R ) 0.047.
There is one molecule per asymmetric unit. The structure was
solved by the direct method using the program SHELXS-86.26
They were refined by the full-matrix least-squares refinement
procedure using the SHELXL package.27 Hydrogen atoms
were also included in the refinement with variable positions
and isotropic temperature factors.
In Vitr o Cytotoxicity Assa ys. MOLT-4, COLO 205, and
HepG2 were obtained from the American Tissue Culture
Collection (ATCC). KB and TSGH 8302 cells were obtained
from the Chinese patients of the Tri-Service General Hospital,
Taipei, Taiwan. COLO 205, KB, and TSGH 8302 cells were
grown as a monolayer in RPMI 1640 (Gibco, BRL) supple-
mented with 5% fetal bovine serum (FBS) (Hyclone). HepG2
cells were grown as a monolayer in IMDM medium (Gibco,
BRL) supplemented with 10% FBS. MOLT-4 was grown as a
suspension culture in RPMI 1640 medium supplemented with
10% FBS and 1% penicillin/streptomycin (Gibco, BRL). Ex-
ponentially growing cell cultures were maintained in a hu-
midified incubator with an atmosphere of 5% CO2-95% air
(NAPCO, Model 5410) at 37 °C. In principle, the assay is
dependent on the cellular reduction of MTT (Sigma Chemical
Co.) to a blue formazan product by the mitochondrial dehy-
Ack n ow led gm en t. This work was supported by
grants from the Development Center for Biotechnology
(No. 05600361 to J .W.C.) and the National Science
Council of the Republic of China (NSC86-2311-B001-
95 to J .W.C. and NSC86-2311-B001-95 to Y.C.L.).
Su p p or tin g In for m a tion Ava ila ble: HMBC spectrum
and X-ray diffraction data for 4g, including crystal data and
structure refinement, atomic coordinates, bond distance, pa-
rameters, and bond angles (7 pages). Ordering information
is given on any current masthead page.
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